CN111410695B

CN111410695B - Chimeric molecule based on autophagy mechanism mediated Tau protein degradation and application thereof

Info

Publication number: CN111410695B
Application number: CN202010240616.1A
Authority: CN
Inventors: 杨爱民; 梅礼刚; 林昌海
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2021-08-31
Anticipated expiration: 2040-03-31
Also published as: CN111410695A

Abstract

The invention discloses a chimeric molecule for mediating Tau protein degradation based on an autophagy mechanism, and the amino acid sequence of the chimeric molecule is shown as SEQ ID NO. 5 or 7. Also disclosed is a nucleic acid molecule encoding the chimeric molecule that mediates Tau protein degradation based on an autophagy mechanism; an expression vector comprising said nucleic acid molecule. Also discloses application of the chimeric molecule, the nucleic acid molecule and the expression vector for mediating the degradation of the Tau protein based on the autophagy mechanism in the degradation of the Tau protein. The invention develops a novel targeted chimeric molecule, and the chimeric molecule is combined on the surface of the Tau of the aggregated protein, so that the Tau protein is recruited into autophagy corpuscles and is promoted to be degraded through an autophagy pathway. The novel protein targeted degradation technology provided by the invention is expected to be applied to different target proteins, so that the defect that the current protein targeted degradation technology based on the ubiquitin-proteasome pathway cannot efficiently degrade intracellular macromolecular components is overcome.

Description

Chimeric molecule based on autophagy mechanism mediated Tau protein degradation and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a chimeric molecule mediating Tau protein degradation based on an autophagy mechanism and application thereof.

Background

Neurodegenerative diseases are a general term for a group of diseases characterized by progressive loss of central neurons, and are of great interest because they are not druggable and seriously affect the quality of life of elderly patients. These include Parkinson's Disease (PD), Alzheimer's Disease (AD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Studies have shown that most neurodegenerative diseases occur with abnormal accumulation of aggregates of misfolded proteins in nerve cells, resulting in toxic effects on neurons.

Aiming at the diseases, the small molecule inhibitor is combined with the active site of the target protein to inhibit the activity of the target protein so as to relieve the symptoms of the neurodegenerative diseases clinically at present. However, long-term use of single small molecule drugs inevitably leads to drug resistance and off-target effects, and the target protein must have an active site for the small molecule drug to bind. This determines that some diseases are still not "druggable".

In recent years, regulation of target protein levels has been achieved at the translational level by protacs (protein Targeting chimeras) protein targeted degradation technologies based on the ubiquitin-proteasome pathway. PROTACs are bifunctional chimeric molecules formed by linking a target protein ligand to a ligand of a specific ubiquitin E3 ligase. The PROTACs protein targeted degradation chimeric molecule can specifically recognize a target protein through a target protein ligand thereof, and recruit E3 through a ligand of ubiquitin E3 ligase, so as to form a ternary complex of the target protein-PROTACs protein targeted degradation molecule-ubiquitin E3 ligase. Ubiquitin E3 ligase further enables the target protein to be ubiquitinated, eventually recognized by the 26S proteasome and degraded. The targeted degradation of various pathogenic proteins is realized by utilizing a PROTACs protein targeted degradation technology, and corresponding medicaments enter clinical tests.

However, proteasomes can only be used to degrade some small-molecule proteins with short half-life, and for protein aggregates with long-life proteins and macromolecules, efficient clearance via ubiquitin-proteasome pathway is difficult. Therefore, the application of the current protein targeted degradation technology in the research fields of degradation of intracellular macromolecular components, neurodegenerative diseases and the like is also greatly limited. The autophagy is a highly conserved protein degradation pathway in eukaryotes, and mainly comprises a double-layer membrane structure which wraps substrates to be degraded (protein aggregates, organelles, pathogenic bacteria and the like) to form autophagosomes, and then the autophagosomes are fused with lysosomes to form autophagosomes, and the substrates to be degraded are degraded under the action of lysosome acid hydrolase. Autophagosome membrane protein LC3 plays an important role in the formation, maturation and substrate recognition of autophagosomes. Studies at the cellular level and in animal models such as mice have shown that the level of intracellular pathogenic β -amyloid, huntingtin (mHTT) and α -Synuclein protein aggregates can be reduced by drug-activated autophagy, thereby effectively alleviating the toxicity of these protein aggregates on nerve cells. The deletion of autophagy-related genes and the inhibition of autophagy can promote the formation of protein aggregates and the toxicity of the protein aggregates to nerve cells. These studies suggest that autophagy may be a useful target for drug development in neurodegenerative diseases. The specific degradation of intracellular macromolecular components is promoted through an autophagy way, and a new idea is expected to be provided for the treatment of neurodegenerative diseases. However, there are currently few studies that utilize the autophagy pathway to target regulatory protein levels.

The microtubule system is a skeletal component of nerve cells and can be involved in a variety of cellular functions. Microtubules are composed of tubulin and microtubule-associated protein, and Tau protein is the most abundant microtubule-associated protein. The biological function of Tau protein in normal brain tissue is to bind tubulin and promote its polymerization to form microtubules; binds to the formed microtubules, maintains microtubule stability, reduces dissociation of tubulin molecules, and induces microtubule bundling. The Tau protein is a phosphorylation modified protein, and Tau protein molecules in normal mature brain tissues contain 2-3 phosphorylation modified groups. And Tau protein in brain tissue of an Alzheimer disease (senile dementia) patient is abnormally over-phosphorylated, 5-9 phosphorylation modifying groups can be contained in each molecule of Tau protein, and normal biological functions are lost. The total amount of Tau protein in brain tissue of patients with Alzheimer's disease is higher than that of normal people, and normal Tau protein is reduced, and abnormal hyperphosphorylated Tau protein is greatly increased. These abnormally phosphorylated Tau proteins accumulate in nerve cells and exert toxic effects on nerve cells, resulting in the deterioration of neuronal functions. The phosphorylated Tau protein is easy to form aggregates, which also causes that the ubiquitin-proteasome pathway is difficult to degrade Tau protein aggregates with high efficiency. Therefore, the development of a new targeted degradation strategy to degrade Tau protein with intracellular dysfunction would be one of the most direct and effective means for treating alzheimer's disease.

Disclosure of Invention

The invention aims to solve the problems and provides a chimeric molecule for mediating Tau protein degradation based on an autophagy mechanism and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a chimeric molecule based on an autophagy mechanism mediated Tau protein degradation, wherein the amino acid sequence of the chimeric molecule is shown as SEQ ID NO. 5 or 7.

A nucleic acid molecule encoding the chimeric molecule that mediates Tau protein degradation based on an autophagy mechanism as described above.

Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO 16 or 18.

An expression vector comprising the nucleic acid molecule described above.

The application of the chimeric molecule for mediating Tau protein degradation based on the autophagy mechanism in degrading Tau protein.

Use of a nucleic acid molecule as described above for degrading Tau protein.

An application of the expression vector in degrading Tau protein.

p62, also known as SQSTM1 protein, is an autophagy substrate and receptor mediating selective autophagy, plays a role in autophagy and apoptosis in tumor cells, and comprises 4 domains, PB1, TB, LIR (LC3 interacting region, LC3 interacting sequence), UBA. Wherein the LIR domain is responsible for binding to the autophagy minibody membrane protein Atg8/LC 3.

The invention constructs a chimeric molecule based on the autophagy mechanism mediated Tau protein degradation by a gene cloning technology. The chimeric molecule consists of Tau protein ligand TTP (Tau targeting peptide) and autophagy minibody membrane protein LC3 interaction sequence LIR, and the principle of mediating Tau protein degradation based on an autophagy mechanism is shown in figure 1.

The study of the chimeric molecule based on the autophagy mechanism mediated Tau protein degradation provided by the invention comprises the following processes: (1) construction of EGFP-Tau and its corresponding mutant EGFP-TauP301L expression vector: with Xho I and EcoR I as enzyme cutting sites, respectively connecting the nucleic acid sequences of Tau and TauP301L proteins to a pEGFP-C1 vector by a gene cloning technology; (2) construction of chimeric molecule mediating Tau degradation (TTP-LIR) expression vector: connecting a nucleic acid sequence of a Tau protein ligand by using Xho I and Hind III as enzyme cutting sites respectively and connecting a nucleic acid sequence of a LIR structure to a pHA-C1 vector by using EcoR I and BamH I as enzyme cutting sites by using a gene cloning technology; (3) co-expression of chimeric molecules mediating Tau degradation with EGFP-Tau or EGFP-TauP301L in cells: the expression vectors of EGFP-Tau and the corresponding mutant EGFP-tauP301L thereof and different chimeric molecule (TTP-LIR) expression vectors mediating Tau degradation are co-transfected into HEK293 cells by transfection reagents; (4) western blot detection of the degradation level of the target protein EGFP-Tau or EGFP-Tau 301L: the protein level of a target protein EGFP-Tau or EGFP-tauP301L is detected by Western blot technology by using EGFP antibody to evaluate the efficiency of TTP-LIR chimeric molecule in mediating Tau protein degradation.

The invention has the beneficial effects that: the invention develops a novel targeted chimeric molecule, when the chimeric molecule is expressed in cells, Tau protein can be identified through a Tau protein ligand, and LIR is combined with autophagy-related protein LC3, so that Tau protein is recruited into autophagy corpuscles, and degradation of the Tau protein through autophagy pathways is promoted. The invention provides a novel protein targeted degradation technology, which is expected to be applied to different target proteins, thereby overcoming the defect that the current protein targeted degradation technology based on ubiquitin-proteasome pathway can not efficiently degrade intracellular macromolecular components.

Drawings

FIG. 1 is a schematic diagram of the mechanism of mediating Tau protein degradation based on autophagy mechanism according to the present invention.

FIG. 2 shows the result of detecting the EGFP-Tau protein degradation efficiency by Western blot, wherein A is the EGFP-Tau degradation efficiency; b is the quantitative analysis of panel a and its significance comparison (in comparison to control, represents P value less than 0.01).

FIG. 3 shows the result of detecting the EGFP-TauP301L protein degradation efficiency by Western blot, wherein A is the degradation efficiency of EGFP-TauP 301L; b is the quantitative analysis of panel a and its significance comparison (P value less than 0.01, P value less than 0.05 compared to control).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are conventional unless otherwise specified.

The main reagent sources used in the invention are as follows:

plasmid pET28 a-Tau: chenyongxiang doctor of Qinghua university present and stored in the laboratory

Plasmids pEGFP-C1, pHA-C1: addgene plasmid Collection of America

Transfection reagent: roche, X-tremagene HP DNA transformation Reagent

PCR amplification premix: 2 XPCR Bestaq^TMMasterMix with dye (G464-dye), Aibiemeng (abm Co., Ltd., Canada)

All restriction endonucleases and T4 ligase were purchased from Takara Bio Inc

Plasmid extraction kit, DNA purification kit, gel recovery kit: beijing kang is a century Biotechnology Co., Ltd

ECL developing solution: shanghai Biyuntian Biotechnology Co., Ltd, Cat number P0018FS

Cocktail protease inhibitors: MedChemexpress (MCE) Inc

0.45 μm PVDF film, GE

The PCR primers in the examples of the present invention were synthesized by Shanghai Bioengineering Co., Ltd.

Example 1

Construction of EGFP-Tau and EGFP-TauP301L fusion protein (target protein) expression vector

(1) PCR amplification of the Tau protein full-length nucleic acid fragment, extracting DNA of a plasmid pET28a-Tau (containing the complete sequence of wild-type Tau protein) as an amplification template: the PCR reaction system is as follows: 2 XPCR Bestaq^TMMasterMix with 12.5. mu.l dye, upstream primer Tau-F1. mu.l,1. mu.l of downstream primer Tau-R, 1. mu.l of DNA template, and 9.5. mu.l of ddH₂O to a final volume of 25. mu.l. The primer sequences are referenced in table 1. The full-length nucleic acid sequence of the wild-type Tau protein is shown in SEQ ID NO. 1.

TABLE 1 Tau protein full-length nucleic acid sequence PCR amplification primers

The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and reaction for 32 cycles; extension is carried out for 10min at 72 ℃, and PCR products are stored at 4 ℃ after the reaction is terminated. And (3) detecting the size of the fragment by agarose gel electrophoresis of the PCR product, and then carrying out gel recovery to obtain the Tau protein full-length nucleic acid fragment.

(2) Enzyme digestion, ligation and plasmid verification: carrying out double enzyme digestion on the obtained Tau protein full-length nucleic acid fragment and pEGFP-C1 carrier plasmid by using restriction endonucleases Xho I and EcoR I, purifying the DNA fragment subjected to enzyme digestion and the carrier fragment by using a gel recovery kit, and connecting the DNA fragment and the carrier fragment by using T4 ligase at 16 ℃ overnight; the ligation product is transformed into DH5 alpha escherichia coli competent cells, coated on an LB solid plate (the final Kan concentration is 50ug/ml), and cultured overnight at 37 ℃; selecting a single clone to carry out colony PCR verification; and extracting the plasmid, sending the plasmid to Shanghai biological engineering Co., Ltd for sequencing, and indicating that the pEGFP-Tau expression vector is successfully constructed if the sequencing is correct.

(3) Construction of EGFP-TauP301L mutant plasmid:

acquisition of the nucleic acid sequence of TauP 301L: through two rounds of PCR reaction, the base at the amino acid site of the wild-type Tau protein P301 is subjected to site-directed mutagenesis, and the full-length nucleic acid sequence of TauP301L is obtained.

The total length of the amino acid sequence of the Tau protein is 441 amino acids, the first round of PCR reaction takes M-F (5'-GGCTCAAAGGATAATATCAAACACGTCCTGGGAGGCGGCAGTGTGCAAAT-3', SEQ ID NO:4) as an upstream primer, takes Tau-R (SEQ ID NO:3) as a downstream primer and takes the total length of the nucleic acid sequence (SEQ ID NO:1) of the wild-type Tau protein as a template to amplify the fragment of Tau291-441, wherein the nucleic acid sequence of the upstream primer M-F is used for protruding P at the 301 siteBecomes L. The PCR reaction system is as follows: 2 XPCR Bestaq^TMMasterMix with dye 12.5. mu.l, upstream primer M-F1. mu.l, downstream primer Tau-R1. mu.l, DNA template 1. mu.l, added with 9.5. mu.l ddH₂O to a final volume of 25. mu.l. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 15s, and reaction for 32 cycles; extension is carried out for 10min at 72 ℃, and PCR products are stored at 4 ℃ after the reaction is terminated.

In the second round of PCR reaction, Tau-F (SEQ ID NO:2) is taken as an upstream primer, the product of the first round of PCR reaction is directly taken as a downstream primer, and the full-length nucleic acid sequence (SEQ ID NO:1) of wild-type Tau protein is taken as a template to carry out PCR reaction. The PCR reaction system is as follows: 2 XPCR Bestaq^TMMasterMix with dye 12.5. mu.l, upstream primer Tau-F1. mu.l, first round PCR product 1. mu.l, template 0.5. mu.l, add 10. mu.l ddH₂O to a final volume of 25. mu.l, the amount of the wild-type Tau protein nucleic acid sequence template should be reduced in this step. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and reaction for 32 cycles; extension is carried out for 10min at 72 ℃, and PCR products are stored at 4 ℃ after the reaction is terminated. After agarose gel electrophoresis determines that the fragment size is correct, the PCR product (i.e., TauP301L nucleic acid fragment) can be used for subsequent enzyme digestion ligation experiments.

pEGFP-C1 is used as a vector framework, and the steps of enzyme digestion, connection, transformation and single colony PCR of the TauP301L nucleic acid fragment and the vector are the same as the construction process of pEGFP-Tau. After the plasmid is verified to be correct by sequencing of Shanghai biological engineering Co., Ltd, the pEGFP-TauP301L expression vector is successfully constructed.

Secondly, construction of TTP-LIR chimeric molecule expression vector (TTP: Tau targeting peptide)

The method is characterized in that pHA-C1 is used as a vector framework, Xho I and Hind III are sequentially used as enzyme cutting sites to be connected with a nucleic acid sequence of a Tau protein ligand at the C terminal through a gene cloning technology, EcoR I and BamH I are used as enzyme cutting sites to be connected with a nucleic acid sequence of an LIR structure interacting with autophagy corpuscle membrane protein LC3, and a TTP-LIR chimeric molecule expression vector (Y185, Y186 and Y208 respectively) mediating Tau protein degradation is constructed. The designed chimeric molecule for targeted degradation of Tau protein consists of Tau protein ligand, Linker and LIR, the amino acid sequence (SEQ ID NO: 5-7) of the corresponding TTP-LIR chimeric molecule in the chimeric molecule expression vector is shown in Table 2, wherein the single underlined part is the Linker sequence, and the double underlined part is the LIR sequence.

TABLE 2 TTP-LIR chimeric molecule amino acid sequences

The specific construction process is as follows:

(1) construction of pHA-TTP plasmid

Firstly, synthesizing a single-chain nucleic acid fragment by Shanghai biological engineering Co., Ltd according to a nucleic acid sequence corresponding to an amino acid sequence of a Tau protein ligand, and introducing viscous ends of Xho I and Hind III enzyme cutting sites at two ends of the sequence to ensure that a double-chain DNA fragment formed by in vitro annealing can be directly connected with a linearized vector fragment. The formed double-stranded DNA fragments comprise TTP nucleic acid sequence and nucleic acid sequence of linker (GSGS), and the sequence design of the single-stranded nucleic acid fragments is shown in Table 3:

TABLE 3 Single-stranded nucleic acid sequence for construction of pHA-TTP expression vectors

Annealing of DNA: synthesizing double-stranded DNA fragments of the amino acid sequence of 'YQQYQDATADEQGGSGS' by annealing the 185-F/R paired nucleic acid sequences in vitro; synthesizing double-stranded DNA fragments of the amino acid sequence of 'KDYEEVGVDSVEGSGS' by in vitro annealing of the nucleic acid sequences paired with 186-F/R; double-stranded DNA fragments of the "ATVIVITLVMLKGSGS" amino acid sequence were synthesized by in vitro annealing of the 208-F/R paired nucleic acid sequences.

And (3) annealing: mu.l of 50. mu.M forward nucleic acid fragment 185-F, 20. mu.l of 50. mu.M reverse nucleic acid fragment 185-R, 10. mu.l of 10 XDNA annealing buffer (100mM TRIS,500mM NaCl,10mM EDTA, pH7.5), and 50. mu.l of nucleic acid free H were added to 500. mu.l centrifuge tubes₂O to a final volume of 100. mu.l. Heating in metal bath at 95 deg.C for 10min, cooling at room temperature, and concentrating nucleic acidAnd (4) measuring the molecular weight for subsequent connection experiments. The in vitro annealing of the 186-F/R and 208-F/R nucleic acid sequences was identical to the annealing step of 185-F/R.

③ connecting the nucleic acid fragment of the Tau protein ligand: pHA-C1 was subjected to double digestion with restriction endonucleases Xho I and Hind III, and the linearized vector fragment was recovered and ligated with the double-stranded DNA fragment of Tau protein ligand annealed and synthesized in step (II) with T4 ligase at 16 ℃ overnight. The ligation product is transformed into DH5 alpha escherichia coli competent cells, coated on an LB solid plate (the final Kan concentration is 50ug/ml), and cultured overnight at 37 ℃; after the PCR verification of the monoclonal colony, plasmid extraction is sent to the sequencing verification of Shanghai biological engineering Co., Ltd, and if the sequencing result is correct, the nucleic acid sequence of the Tau protein ligand is connected with the pHA-C1 vector, namely the construction of the pHA-TTP plasmid is successful.

(2) Construction of pHA-TTP-LIR vector

First, a single-stranded nucleic acid fragment was synthesized by Shanghai Bioengineering Co., Ltd based on a nucleic acid sequence corresponding to the amino acid sequence of LIR. The double-stranded DNA fragment formed by in vitro annealing can be directly connected with the linearized vector fragment by introducing viscous ends of EcoR I and BamH I enzyme cutting sites at two ends of the sequence. The sequence design of the single-stranded nucleic acid fragments is shown in Table 4.

TABLE 4 Single-stranded nucleic acid sequences of LIR Structure

Annealing of DNA: the nucleic acid sequences paired with LIR-F/R were annealed in vitro to synthesize a double-stranded DNA fragment of "SGGDDDWTHLSSKEV" amino acid sequence.

And (3) annealing: mu.l of 50. mu.M forward nucleic acid fragment LIR-F, 20. mu.l of 50. mu.M reverse nucleic acid fragment LIR-R, 10. mu.l of 10 XDNA annealing buffer (100mM TRIS,500mM NaCl,10mM EDTA, pH7.5), and 50. mu.l of nucleic acid free H were added to 500. mu.l centrifuge tubes₂O to a final volume of 100. mu.l. Heating the mixture in a metal bath at 95 ℃ for 10min, cooling the mixture at room temperature, and then measuring the concentration of the nucleic acid for subsequent connection experiments.

③ ligation of LIR nucleic acid fragments: and (2) carrying out double enzyme digestion on the successfully constructed pHA-TTP vector in the step (1) by using restriction endonucleases EcoR I and BamH I, recovering a linearized vector fragment, and connecting the linearized vector fragment with the double-stranded DNA fragment of the LIR structure annealed and synthesized in the step (II) by using T4 ligase at 16 ℃ overnight. The ligation product was transformed into DH 5. alpha. E.coli, spread on LB solid plate (final Kan concentration 50ug/ml) and cultured overnight at 37 ℃; after the PCR verification of the monoclonal colony, extracting the plasmid, and sending the plasmid to a sequencing verification of Shanghai biological engineering Co., Ltd, wherein if the sequencing result is correct, the construction of the TTP-LIR chimeric molecule expression vector pHA-TTP-LIR is successful. The nucleic acid expression sequences of the 3 TTP-LIR chimeric molecules are SEQ ID NO 16 (corresponding to Y185), SEQ ID NO 17 (corresponding to Y186), and SEQ ID NO 18 (corresponding to Y208), respectively.

Third, cell culture and cell transfection

Cell culture:

the cells adopted by the invention are human HEK293 cell lines, and the culture medium is DMEM high-sugar medium (Gibco, cat # C11995500BT) containing 10% fetal bovine serum (NTC special grade, cat # SFBE) and 5% streptomycin double antibody (Gibco, cat # 15140-122). The culture conditions were 5% CO₂And a constant-temperature cell culture box at 37 ℃.

Cell co-transfection:

the pEGFP-Tau expression vector is respectively cotransfected with different TTP-LIR chimeric molecule expression vectors: planting the cells in a 6-well plate in advance and culturing for 12h to ensure complete adherence; each 1ug of DNA was diluted in 100. mu.l of Opti-MEM medium; adding 2. mu.l of transfection reagent to the DNA dilution and allowing to stand at room temperature for 20min to form a transfection complex; the transfection complex was added dropwise to the adherent cell culture dish containing complete medium and the medium was replaced with fresh medium after 24 h.

The cell transfection procedure for pEGFP-TauP301L was the same as that for pEGFP-Tau.

Fourth, detection of target protein degradation level

The lysosome inhibitor Chloroquine (Chloroquine Diphosphate, abbreviated as CQ) treated group was used as a control. After 36h post-transfection, transfected cells were treated with 20 μ M lysosomal inhibitor CQ for 12h, and protein was extracted to detect changes in target protein levels by Western blot. The specific experimental steps are as follows:

(1) discarding the cell culture medium, washing each well of the six-well plate twice with 2ml of precooled PBS, adding RIPA lysate (containing 1mM Cocktail protease inhibitor and PMSF at final concentration) according to the volume of 120 μ l of each well, and performing lysis on ice for 30 min;

(2) scraping cells, transferring cell suspension to 1.5ml centrifuge tube, continuously lysing for 30min on ice, vortexing and shaking for 30s every 10min to fully lyse, and centrifuging at 4 deg.C and 15000rpm for 20 min. The supernatant was transferred to a 1.5ml centrifuge tube and the sample was placed at-80 ℃ for future use or directly used for protein concentration determination. The protein concentration is measured by adopting a BCA method, the OD value of the protein sample under the wavelength of 562nm is measured by an enzyme-labeling instrument, and the concentration of the protein sample is calculated;

(3) western blot detection of Tau protein level

SDS-PAGE: each histone sample was diluted to the same concentration with RIPA lysate and mixed according to 4: 1 and 5 xSDS loading buffer, denaturing at 98 deg.c for 5min, and performing SDS-polyacrylamide gel electrophoresis with concentration of 5% and separation concentration of 12% based on 50ug of each protein sample; and (3) adjusting the voltage to 120V when the protein sample at constant voltage runs to the interface of the concentrated gel and the separation gel, and stopping electrophoresis when the loading buffer runs to the bottom of the gel.

Membrane transfer, blocking and primary antibody incubation: after electrophoresis, the gel was removed and a "sandwich" structure was assembled according to the Bio-Rad wet-spinning apparatus, using 0.45 μm PVDF membrane, which had been activated with methanol before spinning, under the conditions of 250mA for 90 min. After the membrane is transferred, a pre-dyed protein Marker can be seen on the membrane, the membrane is taken out and rinsed once by TBST, and the membrane is placed in 5 percent skim milk (prepared by TBST) and sealed for 1 hour at room temperature. The blocking solution was discarded, TBST rinsed 2 times for 5min each, and the PVDF membrane was incubated overnight at 4 ℃ in primary anti-diluent, and the antibody and primary anti-diluent were mixed at a ratio of 1: dilution at a ratio of 1000. A first antibody: GFP antibody (Mouse), cat No.: sc-9996, available from Santa Cruze Biotechnology; actin antibody (Mouse), cargo number: AF0003, available from Biyuntian Biotechnology corporation; LC3A/B antibody (Rabbit), cat #: 12741, available from Cell Signaling Technology.

Secondary antibody incubation and development: the PVDF membrane was rinsed three times with TBST for 10min each. HRP-labeled secondary antibodies were labeled according to 1: 3000 was diluted with TBST and the PVDF membrane was incubated in a secondary antibody dilution for 1h at room temperature. After the reaction was complete, the PVDF membrane was rinsed three times with TBST for 10min each. According to the weight ratio of the solution A to the solution B, 1: 1, preparing ECL developing solution, uniformly covering the developing solution on the surface of the PVDF membrane, and placing the PVDF membrane on a Bio-rad chemiluminescence imaging system for development and detection. Secondary antibody: HRP-labeled goat anti-mouse IgG (H + L) cat number: a0216, available from Biyuntian biotechnologies; HRP-labeled goat anti-rabbit IgG (H + L), cat #: a0208, available from Biyuntian Biotech.

The results show that among the three selected chimeric molecules targeting Tau protein, Y185, Y208 plasmids had significantly reduced protein levels of EGFP-Tau after co-transfection of HEK293 cells with pEGFP-Tau and were restored under the action of the lysosomal inhibitor Chloroquine (CQ) (results shown in fig. 2A and 2B). The result shows that the chimeric molecule formed by fusion expression of the Tau protein ligand and the LIR can mediate EGFP-Tau to be degraded through an autophagy pathway, namely, the LIR is anchored on the surface of a target substrate Tau protein, and is combined with LC3 protein through the LIR, so that Tau protein is recruited to enter a cell autophagy corpuscle and is efficiently degraded through the autophagy pathway.

In some patients with alzheimer's disease, mutation of P at position 301 of Tau protein is usually accompanied. Mutant Tau p301L protein is more readily phosphorylated than wild-type Tau protein, resulting in dissociation from microtubule structures and thus aggregation in the cytoplasm. Therefore, mutation of Tau protein P301L is one of the causes of Alzheimer's disease, and TauP301L is also frequently used as pathological model Tau protein for in vitro research of Alzheimer's disease pathogenesis. The experimental results show that the protein level of EGFP-TauP301L is also significantly reduced after HEK293 cells are co-transfected with pEGFP-TauP301L by Y185 and Y208 plasmids (the results are shown in FIGS. 3A and 3B). The result shows that the TTP-LIR chimeric molecule provided by the invention also has an obvious degradation effect on the TauP301L protein which is easy to aggregate in cells to cause cytotoxicity, and the Tau protein degradation idea provided by the invention can be used as a strategy for developing and developing medicaments for treating the Alzheimer disease.

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<212> PRT

<213> Artificial sequence

<400> 5

Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln Gly Gly Ser Gly

1 5 10 15

Ser Ser Gly Gly Asp Asp Asp Trp Thr His Leu Ser Ser Lys Glu Val

20 25 30

<210> 6

<211> 31

<212> PRT

<213> Artificial sequence

<400> 6

Lys Asp Tyr Glu Glu Val Gly Val Asp Ser Val Glu Gly Ser Gly Ser

1 5 10 15

Ser Gly Gly Asp Asp Asp Trp Thr His Leu Ser Ser Lys Glu Val

20 25 30

<210> 7

<211> 31

<212> PRT

<213> Artificial sequence

<400> 7

Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Gly Ser Gly Ser

1 5 10 15

Ser Gly Gly Asp Asp Asp Trp Thr His Leu Ser Ser Lys Glu Val

20 25 30

<210> 8

<211> 60

<212> DNA

<213> Artificial sequence

<400> 8

tcgagcttac cagcagtacc aggacgccac cgccgacgag cagggcggca gcggcagcca 60

<210> 9

<211> 60

<212> DNA

<213> Artificial sequence

<400> 9

agcttggctg ccgctgccgc cctgctcgtc ggcggtggcg tcctggtact gctggtaagc 60

<210> 10

<211> 57

<212> DNA

<213> Artificial sequence

<400> 10

tcgagctaag gactacgagg aggtgggcgt ggacagcgtg gagggcagcg gcagcca 57

<210> 11

<211> 57

<212> DNA

<213> Artificial sequence

<400> 11

agcttggctg ccgctgccct ccacgctgtc cacgcccacc tcctcgtagt ccttagc 57

<210> 12

<211> 57

<212> DNA

<213> Artificial sequence

<400> 12

tcgagctgcg acagtgatcg tcatcacctt ggtgatgctg aagggcagcg gcagcca 57

<210> 13

<211> 57

<212> DNA

<213> Artificial sequence

<400> 13

agcttggctg ccgctgccct tcagcatcac caaggtgatg acgatcactg tcgcagc 57

<210> 14

<211> 55

<212> DNA

<213> Artificial sequence

<400> 14

aattcttcag gaggagatga tgactggacc catctgtctt caaaagaagt gtgag 55

<210> 15

<211> 55

<212> DNA

<213> Artificial sequence

<400> 15

gatcctcaca cttcttttga agacagatgg gtccagtcat catctcctcc tgaag 55

<210> 16

<211> 114

<212> DNA

<213> Artificial sequence

<400> 16

taccagcagt accaggacgc caccgccgac gagcagggcg gcagcggcag ccaagcttcg 60

aattcttcag gaggagatga tgactggacc catctgtctt caaaagaagt gtga 114

<210> 17

<211> 111

<212> DNA

<213> Artificial sequence

<400> 17

aaggactacg aggaggtggg cgtggacagc gtggagggca gcggcagcca agcttcgaat 60

tcttcaggag gagatgatga ctggacccat ctgtcttcaa aagaagtgtg a 111

<210> 18

<211> 111

<212> DNA

<213> Artificial sequence

<400> 18

gcgacagtga tcgtcatcac cttggtgatg ctgaagggca gcggcagcca agcttcgaat 60

tcttcaggag gagatgatga ctggacccat ctgtcttcaa aagaagtgtg a 111

Claims

1. A chimeric molecule that mediates Tau protein degradation based on an autophagy mechanism, characterized in that: the amino acid sequence of the chimeric molecule is shown as SEQ ID NO 5 or 7.

2. A nucleic acid molecule, characterized in that: a chimeric molecule encoding the autophagy-based mechanism of claim 1 that mediates Tau protein degradation.

3. The nucleic acid molecule of claim 2, wherein: the nucleotide sequence is shown as SEQ ID NO 16 or 18.

4. An expression vector, characterized in that: comprising the nucleic acid molecule of claim 2 or 3.

5. Use of the chimeric molecule of claim 1 for mediating Tau protein degradation based on autophagy machinery in the preparation of a medicament for degrading Tau protein.

6. Use of the nucleic acid molecule of claim 2 or 3 in the preparation of a medicament for degrading Tau protein.

7. Use of the expression vector of claim 4 in the preparation of a medicament for degrading Tau protein.